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Caffeine in Citrus flowers


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The allocation of purine alkaloids within citrus flowers was studied and found to be linked to anthesis, with 99% of the total flower caffeine confined to the androecium. The main alkaloid is caffeine accompanied by considerable (up to 30% of caffeine) concentrations of theophylline. In the anther, these purine alkaloids reach altogether a concentration of 0.9% dry wt which is close to the caffeine content of the Arabica coffee bean. The pollen alkaloid concentration is in the same range. Much lower but still marked concentrations were found in the nectar. A considerable breakdown of alkaloids during honey production is assumed. The biological significance of this particular secondary compound allocation as well as possible effects on the key pollinator, the honey-bee, are discussed.
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Caeine in Citrus ¯owers
Josef A. Kretschmar, Thomas W. Baumann*
Institute of Plant Biology, University of Zu
Èrich, Zollikerstr. 107, 8008 Zu
Èrich, Switzerland
Received 11 August 1998; received in revised form 4 January 1999; accepted 21 January 1999
The allocation of purine alkaloids within citrus ¯owers was studied and found to be linked to anthesis, with 99% of the total
¯ower caeine con®ned to the androecium. The main alkaloid is caeine accompanied by considerable (up to 30% of caeine)
concentrations of theophylline. In the anther, these purine alkaloids reach altogether a concentration of 0.9% dry wt which is
close to the caeine content of the Arabica coee bean. The pollen alkaloid concentration is in the same range. Much lower but
still marked concentrations were found in the nectar. A considerable breakdown of alkaloids during honey production is
assumed. The biological signi®cance of this particular secondary compound allocation as well as possible eects on the key
pollinator, the honey-bee, are discussed. #1999 Elsevier Science Ltd. All rights reserved.
Keywords: Citrus; Rutaceae; Caeine; Theophylline; Purine alkaloids; Flower development; Nectar; Pollen; Apis mellifera; Honey-bee
1. Introduction
The compilations often found in literature (e.g.
Willaman & Schubert, 1961), regarding the occurrence
of caeine may give the impression that this purine al-
kaloid is shared by a large number of genera.
However, if we rely exclusively on data re-examined by
advanced analytical techniques, we arrive at the con-
clusion that during evolution, 'invention` of caeine,
i.e. the purine alkaloid pathway, was a relatively rare
event meaning that out of ca. 10,000 angiosperm gen-
era only seven developed, to our present knowledge,
this phytochemical feature, namely Coea, Camellia,
Theobroma, Herrania, Cola, Ilex, and Paullinia.
Therefore, a report by Stewart in 1985 (Stewart, 1985)
was most exciting because it claimed the presence of
caeine, even though in the very low range of 6 and
50 ppm (31 and 258 nmol g
fr. wt) in leaves and
¯owers, respectively, of several Citrus species. His ®nd-
ings were con®rmed by an Italian group (Trova,
Cossa, & Gandolfo, 1994) which detected caeine in
dried citrus ¯owers (237 to 856 nmol g
dry wt) com-
mercially available for preparing a tea. Moreover, the
authors found caeine for the ®rst time also in honeys
originating from the activities of honey-bees (Apis mel-
lifera ) visiting either frequently (uni¯oral honey) or
sporadically ('mille®ori` honey) the ¯owers in orange
plantations. Caeine in these honeys ranged from 2.6
to 52 nmol g
. Later, the analyses were extended to
various other citrus honeys (De®lippi, Piancone, &
Tibaldi, 1995; Vacca & Fenu, 1996) and possible
source-¯owers (Vacca, Agabbio, & Fenu, 1997) with
the aim to establish a measure of quality control.
However, a correlation could not yet be established. In
all these studies, neither single ¯ower organs nor nec-
tar and pollen were examined.
In a preliminary investigation on coee (Kretschmar
& Baumann, 1998) we recognised that ¯ower caeine
was relatively abundant (ca. 3200 nmol g
fr. wt) and
preferentially, even though not very markedly, allo-
cated in the androecium together with other purine al-
kaloids. Therefore, we analysed the within-the-¯ower
distribution of these alkaloids in citruses (including
Poncirus ) considered ideal to demonstrate organ-
speci®c allocation because of the low average content.
Indeed, among the ¯ower organs analysed the androe-
cium had by far the highest concentration of alkaloids,
Phytochemistry 52 (1999) 19±23
0031-9422/99/$ - see front matter #1999 Elsevier Science Ltd. All rights reserved.
PII: S0 0 3 1 - 9 4 2 2 ( 9 9 ) 0 0 1 1 9 - 3
* Corresponding author. Fax: +41-1-634-8204.
E-mail address: (T.W. Baumann)
with a main allocation in the anther and pollen at a
very high level. Moderate, but still marked purine al-
kaloid concentrations were found in the nectar. Since
caeine is known to be insecticidal, the results were
discussed also in the context of intoxication of honey-
2. Results and discussion
In a ®rst approach the purine alkaloid content of
entire ¯owers of Citrus paradisi and C. maxima was
determined. Caeine was the main alkaloid (21 and 77
nmol g
fr. wt, respectively) accompanied by theo-
phylline (4 and 15 nmol g
fr. wt, respectively), while
theobromine and paraxanthine occurred in traces only.
Similarly, commercially available orange ¯ower tea (C.
sinensis ) contained caeine (182 nmol g
dry wt) and
theophylline (46 nmol g
dry. wt). These caeine
values are in the range as found before (Stewart, 1985;
Trova et al., 1994), but until now the presence of theo-
phylline and other dimethylxanthines in Citrus has not
been reported. Theophylline is a trace compound in
the `classical' caeine plants consumed by the human.
In some of the citrus ¯ower tissues it showed consider-
able accumulation (Table 1).
The chemical analysis of ¯ower development (C.
limon ) revealed, that the small, round-shaped ¯ower
bud was virtually alkaloid-free, with only a trace of
theophylline, whereas the elongated bud shortly before
anthesis contained well-measurable concentrations of
both caeine and theophylline, accompanied by little
theobromine and paraxanthine. During anthesis the
caeine content increased by a factor of almost 2
(Table 1). Similarly (not shown), two stages (145 and
216 mg fr. wt) of ¯ower buds of the closely related
Poncirus trifoliata were purine alkaloid-free, whereas
the freshly opened ¯ower (375 mg fr. wt) had an over-
all caeine concentration of 109 nmol g
fr. wt. A
similar increase was observed during anthesis of C.
paradisi. Therefore we may conclude that anthesis in
citruses is coordinated with a rapid allocation of caf-
Then, the ¯owers of C. paradisi,C. maxima,,C.
limon and P. trifoliata were separated into petals, pis-
tils and stamens and analysed (the tiny green sepals
and the ¯ower base were found in preliminary exper-
iments to be virtually alkaloid-free). Both, petals and
pistils contained very small concentrations (mostly in
the range of 2 to 10 nmol g
fr. wt, respectively) of
caeine, theobromine, or theophylline (not shown).
However, the stamens (Table 1) contained the highest
concentrations of caeine and theophylline and were
the exclusive site of ¯ower paraxanthine which was
hardly detectable in the related entire ¯owers because
of dilution. The separate analysis of ®lament and
anther revealed maximum alkaloid concentrations in
the latter exceeding altogether the concentration of
Table 1
Purine alkaloid content (nmol g
fr. wt) in ¯owers, nectars and honeys of Citrus spp. n= 3±10 (¯owering units); n.d.=not detectable
Caeine Theobromine Theophylline Paraxanthine
Flower development (C. limon)
Small bud (148 mg; n= 5) n.d. n.d. 621
Large bud (840 mg; n= 3) 16622
Full anthesis (938 mg; n= 3) 318231 27211
C. paradisi 3'233242 28 1732222
C. maxima 1'110213 13 305 245
P. trifoliata 807211 5 56215
C. limon 1'415218 67 115228
C. paradisi 1'917229 18 1392215
C. maxima 850211 8 12 214
C. paradisi 8'5512169 26 1'491229 32
C. maxima 7'9002119 18 2'753 241 20
C. medica 6'8572321 n.d. 1'921290 n.d.
Nectar (nmol ml
C. paradisi 487215 22 552212
C. maxima 912510 3213
C. limon 6022 n.d. n.d. n.d.
Honey (nmol g
Sicilia 31216 3213
California 221 1 n.d. n.d.
Mean value (2the estimated experimental error, see Section 3).
J.A. Kretschmar, T.W. Baumann / Phytochemistry 52 (1999) 19±2320
10,000 nmol (=10 mmol) per g fr. wt. If related to dry
wt, it results a value of ca. 0.7±0.8% caeine and
0.9% total purine alkaloid in the anther. Hence, the
purine alkaloid concentration in the anther is close to
that in the Arabica coee bean (1.2%, almost exclu-
sively caeine)!
Finally, pollen of (due to the absence of blooming
of the other species) Citrus medica (citron) was ana-
lysed. This single analysis revealed a high purine alka-
loid concentration (altogether almost 8800 nmol g
in the microspores (Table 1), which is in the range of
that found in the anther of the closely related species.
We cannot yet decide whether the anther alkaloid
amount is completely con®ned to the pollen, or
whether the anther wall contains alkaloid at concen-
trations similar to pollen. Also, we have not yet stu-
died the localisation of purine alkaloids within the
pollen grain. At the present we can only speculate
about the signi®cance of this conspicuous allocation.
Besides protection against (unknown) pollen predators,
purine alkaloids are well-studied defence compounds
(reviewed in Harborne (1993)), the cytokinin-like eect
of caeine (Vito
Âria & Mazzafera, 1997) may play a
role during pollination and seed set in citrus
(Hernandez Minana & Primo Millo, 1990).
Blossom honey essentially consists of nectar concen-
trated by the activities of specialised bees in the hive.
The pollen present in the honey is quantitatively negli-
gible but a valuable nectar 'contaminant` which is of
help to trace the source ¯owers. It has been reported
that on average 64% of the pollen found in uni¯oral
citrus honeys is citrus pollen (White & Bryant, 1996)
meaning that roughly two third of the honey originate
from citrus nectar. Since the latter was shown to con-
tain caeine in the range of ca. 60 to 490 nmol ml
(Table 1) and undergoes a concentration process by a
factor of ca. 2 during honey production, one should
expect a much higher caeine concentration in citrus
honey than found in the present (2 and 31 nmol g
Table 1) or in earlier (2.5±50 nmol g
(Trova et al.,
1994)) studies. It can be calculated that about 95% of
the nectar caeine is removed or degraded by a still
unknown mechanism. However, even though the nec-
tar was sampled with caution we cannot rule out con-
tamination by pollen.
In order to obtain an estimate of chemical defence
allocation, the number and weight of the individual
¯ower organs were determined and the alkaloid distri-
bution calculated as exempli®ed for C. lemon in Table
2. In summary, one citrus ¯ower contains about 260
nmol (ca. 50 mg) caeine and 300 nmol total purine al-
kaloid, 99 and 96.6%, respectively, allocated to the
androecium! The amount of alkaloids found in the
nectar (ca. 20 ml) at the moment of ¯ower dissection is
negligible in the case of C. lemon (ca. 1 nmol; caeine
only), but was distinctly higher in C. paradisi (ca. 10
nmol) and may be considerable if extrapolated to the
entire period of ¯owering. However, nectar secretion
was not studied in detail. Diurnal ¯uctuations are to
be expected and may account for some of the dier-
ences in pattern and concentrations of nectar purine
alkaloids listed in Table 1.
Finally, we should mention that orange ¯ower tea
(Aurantii ¯os) is pharmaceutically recommended for
treatment of sleeplessness [O
ÈAB, Ph. Helv. VI]. The
amount of caeine ingested by consumption of such a
calming tea is below 100 mg, a dose present in homeo-
pathic coee preparations used against insomnia
(HAB, (Baumann & Seitz, 1992)).
2.1. Caeine and honey-bees
High concentrations of secondary compounds have
been detected in microspores of both wind- (e.g.
Meurer, Wray, Wiermann, & Strack, 1988) and insect-
(reviewed in Detzel & Wink, 1993) pollinated ¯owers.
In the latter case the question of intoxication of the
pollinators arises. Detzel and Wink (Detzel & Wink,
1993) tested a large number of such allelochemicals on
the feeding behaviour of honey-bees. Caeine was
found to act as a deterrent and its toxicity was com-
paratively low under no-choice conditions (LD
at 0.2
%). In an earlier study (Ishay & Paniry, 1979), honey-
bees, oered free choice of either the sugar solution
alone or the sugar solution with the caeine, similarly
preferred the sugar solution. The concentration of the
Table 2
Purine alkaloid allocation within the ¯ower of Citrus lemon. The ¯ower base is virtually alkaloid-free
Organ Caeine aPurine alkaloids
fr. wt (mg) nmol g
fr. wt amount (nmol) % of total nmolg
fr. wt Amount (nmol) % of total
Petals (4±5) 509 3.9 2.0 0.8 15.3 7.8 2.5
Stamens (31±32) 183 1415.0 258.9 99.1 1604.5 293.6 96.5
Pistil (1) 103 2.5 0.3 0.1 29.5 3.0 1.0
Flower base (1) 119 n.d. 0 ± ± ± ±
Total 914 285.8
261.2 100 333.0
304.4 100
Overall concentration of the entire ¯ower. In parenthesis: number of organs per one ¯ower. For nectar see text.
J.A. Kretschmar, T.W. Baumann / Phytochemistry 52 (1999) 19±23 21
caeine solution (ca. 250 mM) happened to be in the
range of citrus nectars (Table 1). It was readily
accepted under no-choice conditions. After ®ve days, a
300±500 % boost in oviposition by the (young) queen,
an enhanced activity of the bees outside the hive, and
an improved defence by bees against hornets at the
hive entrance was observed. In contrast, hornets also
fed with caeine ceased foraging in the ®eld and also
failed to clear the dead (poisoned by caeine) larvae
out of the nest. On the cellular level, caeine was
shown to in¯uence the cytosolic Ca
concentration in
various organisms including honey-bees, where the caf-
feine-sensitive Ca
release from the endoplasmic reti-
culum in photoreceptors has been studied (Walz,
Baumann, & Ciriacy-Wantrup, 1994).
Caeine and related substances are known not only
to exert insecticidal activity but also to synergize the
eects of pesticides (Nathanson, 1984). On the one
side it appears that the toxicity of caeine in the
honey-bee larvae, which to our knowledge do not suf-
fer from the caeine-rich pollen in citrus orchards, is
relatively low. On the other side, however, one should
in future evaluate bee toxicity of pesticides also in the
presence of purine alkaloids, in order to account for
the above-mentioned synergistic eect which may
occur not only in citrus but also in coee and tea plan-
Finally, we should mention that recently citrus pol-
len was found to be toxic to the predatory mite
Euseius mesembrinus (Yue, Childers, & Fouly, 1994)
which is a facultative pollen feeder widely distributed
in citrus plantations. If toxicity is due to the presence
of purine alkaloids, one may suggest that mites, in
contrast to honey-bees, are very susceptible to purine
alkaloids, a situation which could be advantageous in
the chemical control of the ectoparasitic mite Varroa
jacobsoni associated with the honey-bee.
3. Experimental
3.1. Plant material
Citrus plants (C. paradisi Macf., grapefruit; C. max-
ima (Burm.) Merr., shaddock; C. limon (L.) Burm.f.,
lemon, and C. medica L., citron) were grown in the
greenhouse. The trifoliate orange plant (P. trifoliata
Raf.) was kept outdoors in the institute garden. Tea of
orange ¯owers (C. sinensis (L.) Pers. was bought in a
local store (Coop, Switzerland) as well as orange
¯ower honeys of Sicilian (Globus, Switzerland) and
Californian (Biorex AG, Ebnat-Kappel, Switzerland)
origin. Nectar was sampled using a glass (hematokrit)
3.2. Purine alkaloid extraction
Fresh entire ¯owers (n= 3 to 10) or the related
¯ower parts were pooled and extracted in 0.1 N HCl
(1 ml per 50 to 150 mg fr. wt) at 508for 30 min by
sonication. One ml of the extract was applied onto a
Kieselgur column (Extrelut1, Merck). Essential oil was
removed by 12 ml hexane and thereafter purine alka-
loids were eluted with 12 ml CH
. The eluate was
dried under a stream of N
and the residue dissolved,
for HPLC, in 1 ml H
O. Orange ¯ower tea was
extracted likewise (1 ml 0.1 N HCl per 250 mg, with-
out further drying the ¯owers) as well as honey (1 g),
which was diluted with 0.1 N HCl (2 ml) prior to ap-
plication onto the Kieselgur column. Nectar was
mixed (1:1) with 8% MeOH and directly injected.
Pollen was collected and processed as follows: stamens
were harvested, dried at room temp. and transferred
into a pre-weighed Eppendorf tube, which then was
vortexed at high speed to spin o the pollen. The sta-
mens were removed and the weight of the pollen was
determined by weighing the tube again (Mettler AE
240). The pollen (2.84 mg) was suspended in 300 ml 0.1
N HCl. After 3 h at room temp. a 30-min-sonication
at 408followed. Thereafter, the suspension was ®ltered
through a membrane ®lter and directly injected into
HPLC. No attempt was made to determine the dry
weight of the pollen.
3.3. HPLC separation of purine alkaloids
HPLC separation of purine alkaloids was carried
out on a Nucleosil-100-5 C18 HD column; precolumn
48 mm; ChromCart, Macherey-Nagel). Parameters
were controlled by a Hewlett-Packard liquid chromato-
graph equipped with a diode array detector set at 272
nm. Chromatography was carried out using the follow-
ing gradient: 0±4 min with 0±7.5% MeOH and 0±
2.5% AcN, 4±20 min with 7.5% MeOH and 2.5%
AcN. The R
's (min) were 7.1, 9.1, 9.6 and 16.0 for
theobromine, paraxanthine, theophylline, and caeine,
respectively. The ¯ow rate was 1.1 ml min
and injec-
tion vol 150 ml. Peak identi®cation was achieved by
comparing UV spectrum (library established under
separating conditions) and retention time of authentic
3.4. Calculation of the mean value and the `systematic
experimental error'
Since in this study we did not aim at showing the
variability of purine alkaloids and their concentrations
among ¯owers from one or several plants, the ¯owers
were randomly collected and pooled (with ngener-
ally=10, in a few cases of shortage lower but never
less than 3). The obtained mean value has an exper-
J.A. Kretschmar, T.W. Baumann / Phytochemistry 52 (1999) 19±2322
imental error which was calculated by considering the
accuracy of the fresh weight determination (21 mg)
and the quantitation by HPLC (21%).
We thank the beekeepers Kurt Honegger, Baden
and Pierre Pittier, Dietlikon, for answering awkward
questions of botanists and Stefan Bogdanov, Federal
Dairy Research Institute, Bee Department, Bern
Liebefeld, Switzerland, for his expertise in bee-related
topics as well as for critically reading the manuscript.
This work was ®nancially assisted by the Swiss
National Science Foundation, Grant No. 31-50521.97
and by the `Jubila
Èumsspende fu
Èr die Universita
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... mansoanum. Theophylline alkaloids were previously reported in Citrus flower tissues and nectar (Kretschmar and Baumann 1999), as well as in onion nectar (Soto et al. 2016). ...
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Background and aims Nuptial and extranuptial nectaries are involved in interactions with different animal functional groups. Nectar traits involved in pollination mutualisms are well known. However, we know little about those traits involved in other mutualisms, such as ant-plant interactions, especially when both types of nectaries are in the same plant organ, the flower. Here we investigated if when two types of nectaries are exploited by distinct functional groups of floral visitors, even being within the same plant organ, the nectar secreted presents distinct features that fit animal requirements. Methods We compared nectar secretion dynamics, floral visitors, and nectar chemical composition of both nuptial and extranuptial nectaries in natural populations of the liana Amphilophium mansoanum (DC.) L.G.Lohmann (Bignoniaceae). For that we characterised nectar sugar, amino acid, and specialised metabolite composition by HPLC. Key results Nuptial nectaries were visited by three medium- and large-sized bee species and extranuptial nectaries were visited mainly by ants, but also by cockroaches, wasps, and flies. Nuptial and extranuptial nectar differed regarding volume, concentration, milligrams of sugars per flower and secretion dynamics. Nuptial nectar was sucrose-dominated, with high amounts of γ-amino butyric acid and β-amino butyric acid and with theophylline-like alkaloid, which were all exclusive of nuptial nectar. Whereas extranuptial nectar was hexose-rich, had a richer and less variable amino acid chemical profile, with high amounts of serine and alanine amino acids and with higher amounts of the specialised metabolite tyramine. Conclusions The nectar traits from nuptial and extranuptial nectaries differ in energy amount and nutritional value, as well as in neuroactive specialised metabolites. These differences seem to match floral visitors’ requirements, since they exclusively consume one of the two nectar types and may be exerting selective pressures on the composition of the respective resources of interest.
... Previous studies have shown that these phytochemicals, at low doses (25 ppm), improved longevity, pathogen tolerance (Bernklau et al., 2019), and gut microbiomes (Geldert et al., 2021). In addition, other studies suggest that such a dose (25 ppm) is within the range of concentration of phytochemicals in floral nectar (Kretschmar and Baumann, 1999;Palmer-Young et al., 2019). Here, we investigate the impact of the same phytochemicals at the same low dose on HPG size, a physiological trait responsible for brood food production, and discuss our findings in the context of targeted nutritional supplements for healthy honey bees. ...
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Honey bees are the most efficient pollinators of several important fruits, nuts and vegetables and are indispensable for the profitable production of these crops. Health and performance of honey bee colonies have been declining for decades due to a combination of factors including poor nutrition, agrochemicals, pests and diseases. Bees depend on a diversity of plants for nutrition as pollen is the predominant protein and lipid source, and nectar, the source of carbohydrates for larval development. Additionally, pollen and nectar also contain small amounts of plant secondary metabolites or phytochemicals that are primarily plant defense compounds. Bees have coevolved to benefit from these compounds as seen by the improved longevity, pathogen tolerance and gut microbiome abundance in worker bees whose diets were supplemented with select phytochemicals. Here we investigate the impact of four phytochemicals, known to benefit bees, – caffeine, kaempferol, gallic acid and p-coumaric acid, on hypopharyngeal gland (HPG) size of nurse bees. Newly emerged bees were provided with 25 ppm of each of the four phytochemicals in 20% (w/v) sucrose solution and the size of HPGs were measured after a 10 d period. Bees that received p-coumaric acid or kaempferol showed a significant increase in HPG size. A significant decrease in HPG size was seen in bees receiving caffeine or gallic acid. The implication of our findings on worker bee ontogeny, transitioning from nurses to foragers and relevance to foraging related competencies are discussed. It is critical that bees have access to phytochemicals to ensure colony health and performance. Such access could be through natural habitats that provide a diversity of pollen and nectar sources or through dietary supplements for bee colonies.
... Caffeine is hypothesized to have evolved as a deterrent to herbivorous insects (Nathanson 1984), and has been shown at high concentrations (0.01 M) to have negative effects on pollinator behavior Tiedeken et al. 2014). Caffeine, however, is present at low concentrations in the flowers of linden, citrus (10 −5 M) and coffee (10 −4 M) plants (Kretschmar and Baumann 1999;Naef et al. 2004;Maze et al. 2006;Wright et al. 2013) and at these concentrations causes nectar to be preferred by honeybees (Singaravelan et al. 2005), enhances honeybee learning and memory (Si et al. 2005;Wright et al. 2013), and increases bumblebee response to floral odors (Arnold et al. 2021). It has therefore been proposed that low concentrations of caffeine in nectar have positive repercussions for plant pollination, but this has not been studied directly. ...
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Caffeine and ethanol are naturally occurring compounds in floral nectar. We examined how these compounds influenced pollinator behaviors including floral preference, floral constancy, and social behavior using bumblebees, Bombus impatiens, which were given prior experience foraging on either human blue or human white (hereafter blue and white) artificial flowers. Because flower color influenced bee behavior, with strong preferences for blue, we focused on the interaction between nectar chemistry and flower color. Bees that had experience with blue flowers preferred blue regardless of nectar chemistry. In contrast, for bees that had prior experience with white flowers, only the control treatment preferred white, while bees exposed to caffeine and ethanol showed no preference. The effects of nectar compounds may therefore only occur when bees are already foraging on a less-preferred color. We also examined the impact of nectar chemistry on the social behavior of joining other bees at flowers. In the same treatments for which bees showed a preference for previously experienced flower colors (all of the blue treatments and only the white control), bees also preferentially visited unoccupied flowers. In the treatments where bees showed no color preference, however (the white caffeine and ethanol treatments), bees showed no preference for unoccupied flowers. We show that the impacts of field-realistic levels of caffeine and ethanol in nectar on pollinator behavior depend on flower color, highlighting that the potential costs and benefits of nectar chemistry to plants are likely to be dependent on bee behavioral biases for other floral traits. Significance statement Flower nectar often contains toxic compounds hypothesized to impact pollination, but little research has shown their effects on the behavioral decisions of free-flying bees. Caffeine and alcohol occur in the nectar of some flowers. We found that bee response to these nectar compounds depends on the flower color. Bees preferentially visited blue flowers regardless of nectar chemistry, but the presence of caffeine or alcohol reduced bee color preference when bees had experience foraging on white flowers. The bumblebee’s social behavior of joining other bees at flowers showed related effects; in treatments where bees showed a preference for flower type, they also preferred to forage alone. This research highlights that bees make decisions based on the interaction between multimodal cues including nectar chemistry, and therefore the strength of selection on nectar chemistry is dependent on bee behavioral biases for other floral traits.
The biology of citrus is briefly reviewed, pointing up how surprisingly little is known of pollination and dispersal in wild species. The classification of the citron, Citrus medica, in modern science is set out in a historical context: its taxonomic relationships in the light of the evolution of the genus Citrus and the citron's role in the origin of major citrus crops as well as the importance of the etrog citron in the traditional Jewish Tabernacles festival are outlined. The global threat to the citron and all other citrus from the bacterial disease, huanglongbing, is explained. As an aid to understanding the much-confused citrological literature, the formal taxonomy of the citron is presented in an Appendix, complete with a nomenclatural account of those commercial crops which have citron in their make-up; for example, the Rangpur lime (a rough lemon) is Citrus × otaitensis (syn. C. × volcameriana, C. × jambhiri).
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Caffeine has long been used as a stimulant by humans. Although this secondary metabolite is produced by some plants as a mechanism of defense against herbivores, beneficial or detrimental effects of such consumption are usually associated with dose. The Western honey bee, Apis mellifera, can also be exposed to caffeine when foraging at Coffea and Citrus plants, and low doses as are found in the nectar of these plants seem to boost memory learning and ameliorate parasite infection in bees. In this study, we investigated the effects of caffeine consumption on the gut microbiota of honey bees and on susceptibility to bacterial infection. We performed in vivo experiments in which honey bees, deprived of or colonized with their native microbiota, were exposed to nectar-relevant concentrations of caffeine for a week, then challenged with the bacterial pathogen Serratia marcescens. We found that caffeine consumption did not impact the gut microbiota or survival rates of honey bees. Moreover, microbiota-colonized bees exposed to caffeine were more resistant to infection and exhibited increased survival rates compared to microbiota-colonized or microbiota-deprived bees only exposed to the pathogen. Our findings point to an additional benefit of caffeine consumption in honey bee health by protecting against bacterial infections. IMPORTANCE The consumption of caffeine is a remarkable feature of the human diet. Common drinks, such as coffee and tea, contain caffeine as a stimulant. Interestingly, honey bees also seem to like caffeine. They are usually attracted to the low concentrations of caffeine found in nectar and pollen of Coffea plants, and consumption improves learning and memory retention, as well as protects against viruses and fungal parasites. In this study, we expanded these findings by demonstrating that caffeine can improve survival rates of honey bees infected with Serratia marcescens, a bacterial pathogen known to cause sepsis in animals. However, this beneficial effect was only observed when bees were colonized with their native gut microbiota, and caffeine seemed not to directly affect the gut microbiota or survival rates of bees. Our findings suggest a potential synergism between caffeine and gut microbial communities in protection against bacterial pathogens.
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Floral chemical defence strategies remain under-investigated, despite the significance of flowers to plant fitness. We used cyanogenic glycosides (CNglycs)-constitutive secondary metabolites that deter herbivores by releasing hydrogen cyanide, but also play other metabolic roles-to ask whether more apparent floral tissues and those most important for fitness are more defended as predicted by optimal defence theories, and what fine-scale CNglyc localisation reveals about function(s)? Florets of eleven species from the Proteaceae family were dissected to quantitatively compare the distribution of CNglycs within flowers and investigate whether distributions vary with other floral/plant traits. CNglycs were identified and their localisation in florets was revealed by matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI). We identified extremely high CNglyc content in floral tissues of several species (>1% CN), highly tissue-specific CNglyc distributions within florets, and substantial interspecific differences in content distributions, not all consistent with optimal defence hypotheses. Four patterns of within-flower CNglyc allocation were identified: greater tissue-specific allocations to (1) anthers, (2) pedicel (and gynophore), (3) pollen presenter, and (4) a more even distribution among tissues with higher content in pistils. Allocation patterns were not correlated with other floral traits (e.g. colour) or taxonomic relatedness. MALDI-MSI identified differential localisation of two tyrosine-derived CNglycs, demonstrating the importance of visualising metabolite localisation, with the diglycoside proteacin in vascular tissues, and monoglycoside dhurrin across floral tissues. High CNglyc content, and diverse, specific within-flower localisations indicate allocations are adaptive, highlighting the importance of further research into the ecological and metabolic roles of floral CNglycs.
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The identification of unknown chemicals has emerged as a significant issue in untargeted metabolome analysis owing to the limited availability of purified standards for identification; this is a major bottleneck for the accumulation of reusable metabolome data in systems biology. Public resources for discovering and prioritizing the unknowns that should be subject to practical identification, as well as further detailed study of spending costs and the risks of misprediction, are lacking. As such a resource, we released databases, Food-, Plant- and Thing-Metabolome Repository (,, and, referred to as XMRs) in which the sample-specific localization of unknowns detected by liquid chromatography–mass spectrometry in a wide variety of samples can be examined, helping to discover and prioritize the unknowns. A set of application programming interfaces for the XMRs facilitates the use of metabolome data for large-scale analysis and data mining. Several applications of XMRs, including integrated metabolome and genome analyses, are presented. Expanding the concept of XMRs will accelerate the identification of unknowns and increase the discovery of new knowledge.
Acacia saligna is a multi-purpose tree. However, enquiries exist among beekeepers on its importance for honeybees in Tigray region of Ethiopia. The aim of this study was to investigate the effects of A. saligna pollen on Apis mellifera adult worker bees. Feeding trial was conducted on honeybee collected polyfloral pollen (PFP), honeybee collected A. saligna pollen (HCAP) and manually collected A. saligna pollen dust (MCAP) at various level of inclusion. The HCAP and MCAP pollen types were mixed with PFP pollen at a ratio of 1:4, 3:2 and 1:0 to make seven pollen feed treatments. Newly hatched day-old cohort worker bees were collected from one colony and trapped from the sealed brood with a meshed plastic bag before hatching. A ten-day indoor cage experiment was carried out after setting up twenty worker bees per cage. The result showed no statistically significant difference on worker bee mortality and abnormality due to treatment diets, except that higher level of A. saligna pollen inclusion (> 60%) in the diet reduced intake. Secondary metabolites analysis showed higher alkaloids (4.8%) in PFP, total flavonoids of 29.66 mg QE/g in HCAP and 20.61 mg GAE/g phenol in MCAP. The finding implied A.saligna has apicultural value as dry season pollen source rather than considering it as poisonous.
Cytokinin amounts were determined in the ovaries of flowers and fruitlets on leafy and leafless inflorescences of seeded and seedless cultivars of Citrus sinensis, C. reshni and C. clementine. Flowers were sampled over a 65 day period from before anthesis to after fruit set, during the period from fructification to fruitlet expansion. Cytokinin activity appeared in both organic and aqueous phases obtained in the extraction process. Generally, the maximum activity in the organic phase occurred at anthesis, except in ‘Navelate’ orange, and decreased sharply following petal fall, whereas the aqueous phase had more cytokinins later, when the fruitlets had reached 1-2 cm diameter. The effect of pollination on cytokinin-like compounds was studied in seeded cultivars. The pollinated ovaries showed a peak in cytokinin activity at anthesis, in contrast to unpollinated ovaries. Partition chromatography of the organic phase on Sephadex LH-20 indicated five zones of cytokinin activity, four of which had the same elution volumes as ribosyl zeatin, zeatin, isopentenyl adenosine and isopentenyl adenine.
Twenty-nine Sardinian honeys of different botanical origin (Orange, Citrus spp., Asphodel, Cardus spp., Strawberry-tree, Eucalyptus, Multiflowers, Chestnut-tree, Trifolium spp., Edisarum spp., and Mediterranean native flora) currently available on the market, were analyzed in order to determine the caffeine content. The results have shown an average concentration of caffeine of 1.79 and 4.93 mg/ kg in Citrus and Orange honeys respectively. On the contrary, the component was not found in the other honeys. According to previous results obtained by other Authors, these data confirm the peculiarity of Orange and Citrus honeys; therefore their botanical origin can be identified using the caffeine presence and concentration.
Twenty-five samples of different species of citrus flowers (orange, lemon, grapefruit, tangerine, mandarin, mandarin-like and two hybrids) were tested for caffeine content. Although caffeine was found in all samples, the high variability in content within the samples did not allow a precise distinction to be made between the species. Caffeine concentration ranged from 4.61 mg/kg dry matter in a grapefruit sample (cv. "Star Ruby") to 30.92 mg/kg in a tangerine sample (cv. "Clementine di Nules"). The presence of the alkaloid confirms that caffeine found in orange and citrus honeys probably comes from flowers and is transferred to the honey by bees. However, the results of this study do not permit the origin of orange and citrus honeys to be determined from their caffeine content.
Light stimulation of invertebrate microvillar photoreceptors causes a large rapid elevation in Cai, shown previously to modulate the adaptational state of the cells. Cai rises, at least in part, as a result of Ins(1,4,5)P3-induced Ca2+ release from the submicrovillar endoplasmic reticulum (ER). Here, we provide evidence for Ca(2+)-induced Ca2+ release (CICR) in an insect photoreceptor. In situ microphotometric measurements of Ca2+ fluxes across the ER membrane in permeabilized slices of drone bee retina show that (a) caffeine induces Ca2+ release from the ER; (b) caffeine and Ins(1,4,5)P3 open distinct Ca2+ release pathways because only caffeine-induced Ca2+ release is ryanodine sensitive and heparin insensitive, and because caffeine and Ins(1,4,5)P3 have additive effects on the rate of Ca2+ release; (c) Ca2+ itself stimulates release of Ca2+ via a ryanodine-sensitive pathway; and (d) cADPR is ineffective in releasing Ca2+. Microfluorometric intracellular Ca2+ measurements with fluo-3 indicate that caffeine induces a persistent elevation in Cai. Electrophysiological recordings demonstrate that caffeine mimics all aspects of Ca(2+)-mediated facilitation and adaptation in drone photoreceptors. We conclude that the ER in drone photoreceptors contains, in addition to the Ins(1,4,5)P3-sensitive release pathway, a CICR pathway that meets key pharmacological criteria for a ryanodine receptor. Coexpression of both release mechanisms could be required for the production of rapid light-induced Ca2+ elevations, because Ca2+ amplifies its own release through both pathways by a positive feedback. CICR may also mediate the spatial spread of Ca2+ release from the submicrovillar ER toward more remote ER subregions, thereby activating Ca(2+)-sensitive cell processes that are not directly involved in phototransduction.
Hydroxycinnamic acid—spermidine amides have been isolated from pollen and identified from their 1H NMR and mass spectral data: N1,N5-di-(E)-p-coumaroylspermidine from Pterocarya fraxinifolia (Lam.) Spach., N5,N10-di-(E)-feruloylspermidine from Betula verrucosa Ehrh. a mixture of two di-(E)-p-coumaroylspermidines from Alnus glutinosa (L.) Gaertn.
Effects on development, survivorship and reproduction of Euseius mesembrinus (Dean) were studied in the laboratory using nine different plant pollens: ice plant, Malephora crocea (Jacquin); live oak, Quercus virginiana Miller; castor bean, Ricinus communis L.; cattail, Typha latifolia L.; Spanish needle, Bidens pilosa L.; grapefruit, Citrus paradisi (L.) Osbeck; ‘Sunburst’ tangerine, C. reticulata Blanco (hybrid); ‘Hamlin’ orange, C. sinensis (L.) Osbeck; and pummelo, C. grandis (L.) Osbeck. Following eclosion, 100% of adults developed in 7.6 to 7.9 days when pollen of ice plant, live oak, castor bean or cattail was provided as single food sources at 27 ± 1°C. A total of 30.8 and 30.4 eggs per female were obtained when ice plant and live oak pollens, respectively, were provided compared to 23.0 and 19.2 eggs per female when castor bean and cattail pollens were used. Life table parameters were: mean generation time (T) = 12.38; net reproductive rate (Ro) = 17.17; intrinsic rate of natural increase (rm) = 0.2296 and finite rate of increase (λ) = 1.258 when live oak pollen was used as the food source compared with T = 12.47, Ro = 17.79, rm = 0.2308 and λ = 1.260 for ice plant pollen. Castor bean and cattail pollens are available as potential food sources for E. mesembrinus, but they are not as effective as ice plant or live oak pollens. Larvae of E. mesembrinus required 8.5 days to develop to adults, while only 72% of the larvae survived when B. pilosa pollen was used as the food source. Each female deposited an average of 0.45 eggs per day during the first 5 days of oviposition. Citrus pollens including C. paradisi, C. reticulata, C. sinensis and C. grandis are available as food sources for E. mesembrinus, and survivorship ranged from 64 to 84%. Developmental time was prolonged (8.8 to 12.5 days) and fecundity was also very low (0.10 to 0.67 eggs/female/day) during the first 5 days of oviposition compared with both ice plant and live oak pollens.
Methyl anthranilate (MA) contents of 159 samples of Florida honey from 10 crop years are summarized (mean, 2.79; range, 0−5.04 ppm). A highly significant relationship between MA and citrus pollen content is shown for 85 samples from 2 crop years. Sixty-three samples of “monofloral” Florida citrus honeys from two crops averaged 64% citrus pollen and 3.1 ppm of MA. Keywords: Citrus honey; methyl anthranilate; pollen analysis
Caffeine was isolated and identified in extracts from flower buds of several citrus cultivars and from leaves of Valencia oranges (Citrus sinensis L. Osbeck). No caffeine was detected in orange juice. Identification was by high-performance liquid chromatography (HPLC), gas chromatography (GC), ultraviolet spectroscopy (UV), and mass spectra (MS).